2.1. SHIGA TOXIN
Shiga toxin (ST) is a multimeric protein toxin produced by the bacterium Shigella dysenteriae type I. It was first described in 1903 in the prototypic species, Shigella dysenteriae type I (Conradi, Dtsch. Med. Wochenschr. 20:26-28 (1903)). Parenteral injection of ST into susceptible animals results in a delayed limb paralysis followed by death (Keusch et al., 1982, Pharmacol. Ther. 15a:402-435). ST is thought to play a significant role in the pathogenesis of Shigellosis (Keusch et al., 1982, supra; Keusch et al., 1985, Ciba Foundation Symp. 112:193-214). ST has multiple biological activities including enterotoxicity, neurotoxicity, and cytotoxicity (Keusch et al., 1982, supra) and is a potent inhibitor of protein synthesis through the inactivation of the 60S ribosomal subunit (Reisbig, et al., 1981, J. Biol. Chem. 2566:8739-8749).
A molecule of ST consists of one 32 kDa polypeptide chain (termed the A chain) and five 7.6 kDa polypeptide chains (termed the B chain) (Donohue-Rolfe et al., J. Exp. Med. 160:1767-1781 (1984); Donohue-Rolfe et al., Molec. Microbiol. 3:1231-1236 (1989)) The A chain is responsible for the ST's ability to inhibit protein synthesis (Igarashi et al., 1987, FEMS Microbiol. Lett. 44:91-94). Jacewicz et al. (1986, J. Exp. Med. 163:1391-1404) demonstrated that ST binds specifically via its B chain subunit to a glycolipid present in both rabbit jejunal mucosa and in human HeLa cells. This receptor has been identified as globotriaosylceramide (Gb3), Gal.alpha.l.fwdarw.4Gal.beta.l.fwdarw.4Glc.beta.l-Ceramide. ST also binds to the P1 blood group antigen present in human erythrocyte glycolipid extracts. Common to both Gb3 and P1 antigen is a terminal Gal.alpha.l-4Gal disaccharide.
Over the last decade, ST has been purified to homogeneity (reviewed in Keusch et al., Methods In Enzymology 165:152-162, 399-401 (1988)). Examples of purification methods include gel-exclusion chromatography, antibody affinity chromatography, chromatofocusing, and/or Blue Sepharose chromatography. ST has most commonly been detected by: its lethality in vivo, using LD.sub.50 measurements; its enterotoxic activity using measurement of fluid secretion in rabbit intestine (Keusch et al., 1972, J. Clin. Invest. 51:1212-1218); or cytotoxic activity measuring tissue culture LD.sub.50 (Keusch et al., 1972, J. Infect. Dis. 125:539-541). Recently, the present inventors developed a method for detecting ST using an ELISA assay, employing a mouse monoclonal antibody (mAb) specific for the ST B subunit and a rabbit polyclonal antibody specific for the holotoxin (Donohue-Rolfe et al., 1986, J. Clin. Microbiol. 24:65-68). Though the ELISA method is sensitive, to 12 pg of toxin/well, it is somewhat tedious, requiring two types of antibodies.
2.2. SHIGA-LIKE TOXINS I AND II
Cytotoxins with biological properties similar to ST have been identified in a variety of bacterial species, including E. coli, Vibrio, Salmonella, and Campylobacter species (O'Brien et al., 1987, Microbiol. Rev. 151:206-220). In 1977, an E. coli toxin, designated "Verotoxin", was reported to be cytotoxic to Vero cells and distinct from the well-known heat labile and heat stable E. coli toxins (Konowalchuk, 1977, Infect. Immun. 18:775-779). This toxin and the E. coli strains producing it are associated with the diseases of hemolytic uremic syndrome and hemorrhagic colitis (Riley et al., 1983, N. Engl. J. Med. 308:681-685).
Because the cytotoxin produced by E. coli 0157:H7, a strain associated with hemorrhagic colitis, was neutralized by an antiserum raised against Shiga toxin, it was designated "Shiga-like toxin" (O'Brien et al. 1983, Lancet i:702-703). Further studies of E. coli 0157:H7 strain 933 revealed two toxin converting phages termed 933J and 933W (O'Brien et al., 1984, Science, 226:694-696). E. coli 0157:H7 produced two cytotoxins, only one of which could be neutralized by antisera to ST (Scotland et al., 1985, Letter, Lancet ii:885-886). These toxins have been designated Shiga-like toxin I and II (SLT-I and SLT-II) respectively (Strockbine et al., 1988, J. Bacteriol. 170:1116-1122).
SLT-I encoded by phage 933J predominates in E. coli 0157:H7 cell lysates, whereas SLT-II encoded by phage 933W predominates in culture supernatants (Strockbine et al., supra). SLT-I and SLT-II share the biological activities of neurotoxicity in mice, enterotoxicity in ligated rabbit ileal segments and cytotoxicity to both Vero and HeLa cells (Strockbine et al., supra). ST, SLT-I and SLT-II all inhibit protein synthesis by inactivating the 60S ribosomal subunit. All three toxins cleave the N-glycosidic bond at A-4324 in 28S ribosomal RNA (Endo et al., 1988, Eur. J. Biochem. 171:45-50), thus indicating conservation of the active enzymatic site in the A subunits of these toxins.
Nucleotide sequencing of the toxin genes has shown that the mature ST and SLT-I proteins differ by only one amino acid substitution in the A subunit (Strockbine, 1988, supra). SLT-I and SLT-II, however, are only 58% homologous at the nucleotide level and 56% homologous at the amino acid level (55% A subunit homology and 57% B subunit homology) (Jackson et al., 1987, FEMS Microbiol. Lett. 44:109-114). SLT-II consists of A and B subunits, although its precise subunit stoichiometry has not been clearly defined.
Despite the common functional attributes of ST, SLT-I and SLT-II, the lack of neutralizing immunologic cross-reactivity has resulted in a separation into two groups (ST and SLT-I vs. SLT-II) (Perera et al., 1988, J. Clin. Microbiol. 26:2127-2131 and Pouch-Downes et al., 1988, Infect. Immun. 56:1926-1933). Since ST, SLT-I and SLT-II bind to the same receptor, and since the toxins' binding domain is exposed on the surface, an antibody directed against the binding domain of one of these toxins would be expected to cross-react with the other related molecules. Similarly, the identical and highly specific mechanism of action of the A subunits of the three toxins predicts a conserved sequence of the active enzymatic site which could induce cross-reactive antibodies. These features make the initially reported apparent lack of immunological cross-reactivity between ST/SLT-I and SLT-II puzzling. Recently, a mAb against SLT-II was found to cross-neutralize SLT-I from E. coli strain H30 (Donohue-Rolfe et al., Infec. Immun. 57:3888-3893 (1989)).
As more members of the SLT family have been described and new nomenclature proposed, the field has become increasingly complex and confused. It is now clear that certain E. coli strains contain a second toxin operon which encodes a toxin that is closely related to SLT-II, which has been designated SLT-IIc (Schmitt, C. K. et al., Infect. Immun. 59:1065-1073 (1991)). Oku, Y. et al., Microb. Path. 6:113-122 (1989) had described an SLT from a human clinical isolate of E. coli 91:H21, strain B2F1, which, while immunologically related to SLT-II, was significantly more cytotoxic to Vero cells than to HeLa cells. This SLT was therefore designated an SLT-II variant of human origin (SLT-IIvh). Subsequently Ito, H. et al., Microb. Pathog. 8:47-60 (1990), found that E. coli 091:H21, strain B2F1, contained two SLT-II toxin operons, one of which was very similar to SLT-IIc described by Schmitt et al., (supra). Gannon, V.P.J. et al., J. Gen. Microb. 136:1125-1135 (1990), also isolated an SLT-IIvh, designated SLT-IIva which was closely related to an SLT-II variant of porcine origin.
At least one SLT, associated with porcine disease (edema disease of swine), is biologically very similar to the other SLT-II toxins, was not neutralized by antisera to ST and SLT-I and was reported to be more cytotoxic to Vero cells than to HeLa cells. This toxin has been designated SLT-II variant-porcine (SLT-IIvp) (Marques, L.R.M. et al., FEMS Micro. Lett. 44:33-38 (1987); Weinstein, D. L. et al., J. Bacteriol 170:4223-4230 (1988)).
Differences in cytotoxicity of these toxins for different target cell types may be related to their differential binding to different glycolipid receptors. ST, SLT-I and SLT-II bind preferentially to Gb3, whereas SLT-IIvp, the porcine edema toxin, binds preferentially to globotetraosylceramide (Gb4), although it also binds to Gb3 (DeGrandis, S. et al., J. Biol. Chem. 264:12520-12525 (1989); Samuel , J. E. et al., Infect. Immun. 58:611-618 (1990)). The common binding moiety for SLT's, the a1.fwdarw.4 linked galactose disaccharide, is terminal in Gb3 (Gal.alpha.1.fwdarw.Gal.beta.1.fwdarw.4Glc-Ceramide) whereas it is internal in Gb4 (GalNAc.beta.1.fwdarw.3Gal.alpha.1.fwdarw.4Gal.beta.1.fwdarw.4Glc-Ceramide ). Samuel et al. (supra) found that SLT-IIvh isolated from strain B2F1 bound strongly to galabiosylceramide, Ga2 (Gal.alpha.1.fwdarw.4Gal.beta.1--1Ceramide), and to Gb3, but not to larger glycolipids.
2.3. P1 GLYCOPROTEIN
The P1 glycolipid antigen in human erythrocytes forms part of the P blood group system described many years ago by Landsteiner and Levine (Landsteiner et al., 1927, Proc. Soc. Exp. Biol. Med. 24:941-942). Th P system is now known to consist of three antigens (P1, P and Pk) and at least five phenotypes (P1, P2, P, P1K and P2k) (Marcus, 1981, Sem. Hematol. 18:6371). In 1957 it was reported that material from Echinococcus granulosus hydatid cysts, later identified as a glycoprotein (Morgan, 1964, Proc. 9th Cong. Int. Soc. Blood Transf., Mexico, 1962, pp. 225-229; Cameron et al., 1957, Nature 179:147-148), exhibited P1 blood group reactivity and was a competitive inhibitor of the hemagglutination of P1 positive erythrocytes by typing antisera. Thus, the cysts containing live scolices of the tapeworm Echinococcus granulosus provide a relatively convenient source of soluble P1-active substance. The P1gp antigenic determinant was subsequently shown to be a trisaccharide (Gal.alpha.1.fwdarw.4Gal.beta.1.fwdarw.4GlcNac) identical to the non-reducing end of the P1 glycolipid on human erythrocytes (Cory et al., 1974, Biochem. Biophys. Res. Comm. 61:1289-1296).